This study will elucidate the factors critical in regulating the sialic acid content of a mucin-type glycoprotein. Sialic acid content has been correlated with metastatic ability of tumor cells, and sialic-acid-rich mucins have been shown to mask cell-surface antigens in mouse mammary tumor cells. Sialic acid metabolism is being investigated in MAT-B1 and MAT-C1 rat mammary tumor (13762) ascites sublines, which differ in xenotransplantability and in sialic acid content of the major mucin-type glycoprotein. Three types of studies have been performed. First, characterization of sialyltransferase and CMP-sialic acid synthetase demonstrated that there was no difference in the activity or specificity of these enzymes between MAT-B1 and MAT-C1 preparations. Second, sialic acid analysis revealed that the difference in sialic acid content between the cell lines could be accounted for by N-glycolylneuraminic acid (NeuG1), since MAT-B1 (nontransplantable) cells were devoid of NeuG1 whereas MAT-C1 (xenotransplantable) cells contained equal amounts of N-acetylneuraminic acid (NeuAc) and NeuG1. Structural analysis of MAT-C1 oligosaccharides indicated that the two types of sialic acid were not randomly distributed, but that in monosialylated oligosaccharides NeuG1 was preferentially associated with the Gal(beta1-4)G1cNAc terminus of the core tetrasaccharide Gal(beta1-4)G1cNAc(beta1-6)(Gal(beta1-3))GalNAc. Third, incorporation of label from [3H]-N-acetylmannosamine into free, activated, and bound NeuAc and NeuG1 was monitored for MAT-B1 and MAT-C1 cells. MAT-B1 cells did not synthesize free NeuG1 or CMP-NeuG1. Thus, the enzyme N-acetylneuraminate monooxygenase, which converts NeuAc to NeuG1, is probably deficient in MAT-B1 cells. Studies of the monooxygenase in MAT-C1 cells are currently in progress. It appears that regulation of not only sialic acid content but also the type of sialic acid present may be critical to phenomena such as metastasis and recognition by the immune system. (A)